White adipose tissue (WAT) is the main lipid reservoir that becomes dysfunctional in obesity. It is important to understand how breached lipid homeostasis leads to metabolic disease. Efficient long chain fatty acid (FA) and cholesterol trafficking depends on a transmembrane transporter protein CD36. However, the mechanism through which CD36 switches from uptake to mobilization of lipids and how lipids traffic between the endothelium and adipocytes is not understood. We had previously identified a WAT-specific plasmalemmal interaction between prohibitin-1 (PHB) and annexin A2 (ANX2). To study its role on the surface of endothelial cells (EC) and adipocytes, we have been supported by grant R01DK088131 from the NIDDK. We have shown that PHB/ANX2 binding in the context of lipid rafts mediates FA transport from EC to adipocytes, hence promoting adipocyte lipid deposition. As we recently reported, PHB and ANX2 interact with CD36 on the cell membrane within a complex assembled in response to extracellular FA. Our preliminary data suggest that FA also trigger interaction of PHB and CD36 on the surface of tumor cells, in which function of these proteins has been independently linked to cancer aggressiveness. In this competing renewal application, we propose to investigate the assembly of the PHB/ANX2/CD36 complex and its role in lipid import and export in WAT endothelium, adipocytes, and cancer cells. Our underlying hypothesis is that a spike in either extracellular or intracellular FA brings together PHB, ANX2 and CD36, which then cooperate in either FA uptake or FA mobilization, respectively. By using mice and cell culture models in which PHB or CD36 are deleted in endothelial cells or adipocytes, we will test if these proteins are necessary for lipid import into adipocytes (Specific Aim 1) and for lipolysis-induced lipid mobilization from WAT (Specific Aim 2). Our collaborator, Dr. Maria Febbraio, has generated a mouse model for tissue-specific CD36 deletion, and preliminary data demonstrate CD36 importance in EC for FA and cholesterol homeostasis. We will also use the inter-cellular lipid transport assay that we have designed to study lipid exchange between EC and adipocytes and the function of PHB/ANX2/CD36 interaction in lipogenic and lipolytic conditions. By using cell culture systems in which the protein interaction is disrupted either genetically or pharmacologically, we will also test the function of the PHB/ANX2/CD36 complex in lipid transport from WAT to malignant cells and in cancer chemoresistance and progression (Specific Aim 3). Finally, based on the reported requirement of CD36 palmitoylation for its localization to lipid rafts and for activation of lipid transport, we hypothesize that S-acylation is the trigger of the PHB/ANX2/CD36 complex assembly. Specifically, based on our preliminary data, we hypothesize that CD36 S-acylation occurs upon extracellular or intracellular FA concentration increase. We will collaborate with Dr. Askar Akimzhanov, an expert in protein S-acylation, to test if this process enables FA-induced assembly of the PHB/ANX2/CD36 complex to serve as a switch for context-dependent lipid uptake or mobilization.